Use of an rxr agonist in treating drug resistant her2+ cancers

ABSTRACT

The present specification provides combinations of active agents for the improved treatment of Her2 +  cancers and associated methods of treatments. The combinations comprise an RXR agonist and a Her2-targeted therapeutic agent and may optionally further comprise thyroid hormone.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication 63/286,981 filed Dec. 7, 2021, which is incorporated byreference herein in its entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under grant numberHHSN261201500018I awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

Compounds which have retinoid-like biological activity are well known inthe art and are described in numerous United States patents including,but not limited to, U.S. Pat. Nos. 5,466,861; 5,675,033 and 5,917,082,all of which are herein incorporated by reference. Preclinical studiessuggest that selective activation of Retinoid X Receptors (RXR), whichmodulate functions associated with differentiation, inhibition of cellgrowth, apoptosis and metastasis, with RXR-acting agents (rexinoids) maybe useful in treating a variety of diseases associated with thebiochemical functions modulated by RXR.

For example, TARGRETIN® (bexarotene), which is a RXR agonist havingretinoic acid receptor (RAR) agonist activity as well, was approved bythe U.S. Food and Drug Administration for the treatment, both oral andtopical, of cutaneous manifestations of cutaneous T cell lymphoma inpatients who are refractory to at least one prior systemic therapy.Encouraging results were obtained with TARGRETIN® in several Phase IIstudies in NSCLC (non-small cell lung cancer). However, the pivotalPhase III clinical study did not show increased survival. One possibleexplanation for the limited success of bexarotene is that its activationof RAR decreases its efficacy as an anticancer agent. Thus, moreselective RXR agonists may hold greater promise.

Agents targeting human epidermal growth factor receptor 2 (Her2), bothanti-Her2 antibodies and inhibitors of the tyrosine kinase activity ofHer2, have had significant, but not universal, success in treating Her2⁺cancers, particularly Her2⁺ breast cancers.

Treatments for cancer are ever evolving, gaining in specificity andsophistication. Early non-surgical cancer treatments generally targetedrapidly dividing cells which were more sensitive to radiological andchemical assault. Over time, more specific and less generally toxictreatments have been developed. Some treatments appear to have broadapplicability, for example immune checkpoint inhibitors or rexinoids.Others are targeted to cancers that express a particular antigen orother biomarker involved in the regulation of proliferation ordifferentiation; including many monoclonal antibodies and kinaseinhibitors. Yet as the variety of cancer treatments has grown, it hasbecome ever harder to determine which treatments might be productivelycombined and for what indications.

SUMMARY

Herein disclosed are improved methods of treatment of Her2⁺ cancers,particularly Her2⁺ cancers that are resistant to a Her2-targetedtherapeutic agent. These methods comprise treating a patient having aHer2⁺ tumor with a combination of a Her2-targeting therapeutic agent anda RXR agonist capable of inhibiting cancer growth. In some embodiments,the cancer is also resistant to the RXR agonist. In some embodiments thetreatment combination further comprises thyroid hormone.

In some embodiments, the RXR agonist is capable of activating RXR/Nurr1heterodimeric receptors. In some embodiments the RXR agonist is acompound of Formula I as disclosed herein below, or apharmaceutically-acceptable salt thereof. In some embodiments the RXRagonist is a compound of Formula II as disclosed herein below, or apharmaceutically-acceptable salt thereof. In some embodiments, compoundsof Formula I and Formula II, and their pharmaceutically-acceptablesalts, are referred to as means for activating RXR/Nurr1 heterodimericreceptors or rexinoid means for inhibiting tumor growth.

In some embodiments, Her2-targeting therapeutic agent is an inhibitor ofHer2 kinase activity or Her2-mediated signaling. In some embodimentsHer2-targeting therapeutic agent is therapeutic anti-Her2 antibody.Therapeutic antibodies may mediate antibody-dependent cellularcytotoxicity (ADCC) instead of, or in addition to, inhibiting signaling(kinase activity). Trastuzumab and pertuzumab are examples oftherapeutic anti-Her2 antibodies, as disclosed herein below. In someembodiments such antibodies are referred to as immunoglobulin means forinhibiting Her2⁺ tumor cell proliferation, means for mediating ADCC ofHer2⁺ tumor cells, or immunoglobulin means for inhibiting Her2signaling.

In some embodiments, a Her2-targeting therapeutic agent is anantibody-drug conjugate comprising an anti-Her2 antibody. In someembodiments, the anti-Her2 antibody has therapeutic activity alone,while in other embodiments it does not, merely serving to deliver acytotoxic agent to Her2⁺ cells. Ado-trastuzumab emtansine is an exampleof a Her2-targeting antibody-drug conjugate, as disclosed herein below.In some embodiments such antibody-drug conjugates are referred to asmeans for delivering a cytotoxic agent to Her2⁺ cells.

In some embodiments, the inhibitor of Her2 kinase activity orHer2-mediated signaling is a small organic molecule (small drug)inhibitor of Her2 kinase activity. Lapatinib, neratinib, and tucatinibare examples of Her2 kinase inhibitors as disclosed herein below. Insome embodiments such small drug inhibitor of Her2 kinase activity arereferred to as small molecule means for inhibiting Her2 kinase activity.

In some embodiments, the Her2⁺ cancer is a Her2⁺ breast cancer. In someembodiments the Her2⁺ cancer is a Her2⁺ ovarian cancer, stomach cancer,adenocarcinoma of the lung, uterine cancer (such as serous endometrialcarcinoma), gastric cancer, or salivary duct carcinoma.

In some embodiments, tumor cell growth is inhibited by the combinationof the Her2-targeted therapeutic and the RXR agonist, and the inhibitionof tumor cell growth by the combination is greater than additiveinhibitory effects of each of the Her2-targeted therapeutic and the RXRagonist alone.

In some embodiments, the herein disclosed treatments are carried outconcurrently with other pharmaceutical therapies or radiotherapies. Inalternative embodiments, the herein disclosed treatments are theexclusive therapy in the time interval in which they are conducted. Insome embodiments, the herein disclosed treatments serve as a debulkingtreatment in preparation for subsequent surgical removal of tumor. Insome embodiments, the herein disclosed treatments are applied asadjuvant therapy subsequent to surgical removal of tumor to address anyresidual disease or potential recurrent disease.

Other embodiments include combination drug compositions or formulationscomprising at least one Her2-targeting therapeutic agent and at leastone RXR agonist capable of inhibiting cancer growth. In otherembodiments the combination (used for treatment) may include more thanone agent in one or the other classification (Her2 targeting agentand/or RXR agonist). In still other embodiments the combination mayfurther include thyroid hormone. In some embodiments the thyroid hormoneis thyroxine.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-B are three-dimensional plots depicting the growth inhibitoryeffects of IRX4204 and trastuzumab, alone and in combination, on breastcancer cell lines: MCF7 cells (FIG. 1A) and SkBr3 cells (FIG. 1B).

FIGS. 2A-D are three-dimensional plots depicting the growth inhibitoryeffects of IRX4204 and lapatinib, alone and in combination, on breastcancer cell lines: MCF7 cells (FIG. 2A), SkBr3 cells (FIG. 2B), BT474cells (FIG. 2C), and MDA-MB-361 cells (FIG. 2D).

FIGS. 3A-D are three-dimensional plots depicting the growth inhibitoryeffect of IRX4204 and neratinib, alone and in combination, on breastcancer cell lines: MCF7 cells (FIG. 3A), SkBr3 cells (FIG. 3B), BT474cells (FIG. 3C), and MDA-MB-361 cells (FIG. 3D).

FIGS. 4A-B depict the growth inhibitory effect of IRX4204 and neratinib,alone and in combination, on Her2⁺ breast cancer cell line HCC1954,which is resistant to both agents individually. FIG. 4A is athree-dimensional plot showing the growth inhibitory effect for a matrixof various concentrations of the two agents. FIG. 4B presents linearplots of the same data for no and 1000 nM IRX4204 for the variousconcentrations of neratinib.

FIG. 5A-B are three-dimensional plots depicting the growth inhibitoryeffect of IRX4204 and tucatinib (FIG. 5A) or lapatinib (FIG. 5B) aloneand in combination, on Her2⁺ breast cancer cell line HCC1954, which isresistant to each of the agents individually.

DESCRIPTION

The herein disclosed embodiments include methods of treating Her2⁺cancers that are resistant to Her2-targeted therapeutic agents, such asresistant Her2⁺ carcinoma of the breast, with a combination a retinoid Xreceptor (RXR) agonist (rexinoid) and a Her2-targeted anticancer agent.The Her2⁺ cancers resistant to Her2-targeted therapeutic agents may alsobe resistant to the RXR agonist. Some embodiments further compriseadministration of thyroid hormone in conjunction with the RXR agonist.Embodiments include a RXR agonist for use in combination with aHer2-targeted anticancer agent, or a thyroid hormone and a Her2-targetedanticancer agent, in the treatment of Her2⁺ cancers resistant toHer2-targeted therapeutic agents. In some embodiments, the Her2⁺ canceris Her2⁺ breast cancer.

Even in light of the usefulness of combinations of Her2-targetedtherapeutic agents and the disclosed RXR agonists in treating Her2⁺cancers that are sensitive to Her2-targeted therapeutic agents, it isremarkable that the combination of a Her2-targeted therapeutic agent andthe disclosed RXR agonists can be effective against Her2⁺ cancers thatare resistant to the Her2-targeted therapeutic agent. Moreover, atappropriate concentrations of the therapeutic agents, the effect ofthese combinations is more than additive (that is, synergistic). It iseven more remarkable that this holds true when the cancer is alsoresistant to the RXR agonist.

Three small molecule drugs that inhibit Her2 kinase activity, but arenot close structural analogs of each other, neratinib, lapatinib, andtucatinib, were tested for growth inhibitory effect on a Her2⁺ breastcancer cell line in combination with the RXR agonist IRX4204.Synergistic growth inhibition was observed for all three kinaseinhibitors with IRX4204. Thus, there is an apparent class effect withrespect to inhibitors of Her2 kinase activity. In some embodiments, theinhibitor of Her2 kinase activity is a small organic molecule. In someembodiments, the inhibitor of Her2 kinase activity is an antibody, forexample, trastuzumab (HERCEPTIN®), margetuximab (MARGENZA®), orpertuzumab (PERJETA®).

The methods of treatment disclosed herein involve the administration ofa combination of two, three, or more therapeutic agents. Moreover, theadministration of one of these agents may be described as being done incoordination or conjunction with another of these agents. By suchcombination, or administration of in coordination or in conjunctionwith, it is meant that the manner of administration of each of theseagents is such that the physiologic effects of the agents overlap intime. This does not require that the agents be contained in the samecomposition or formulation, nor that they be administered as separatecompositions at the same time, by the same route of administration, oron the same schedule, though in some embodiments any of the foregoingmay be the case. For example, while it is possible to administer RXRagonists, thyroid hormone, and Her2 kinase inhibitors on a dailyschedule, antibodies are more typically administered at intervalsmeasured in weeks.

Among the earlier successful targeted cancer therapies are thosetargeting human epidermal growth factor receptor 2 (Her2). Her2-targetedtreatments include monoclonal antibodies (mAbs), such as trastuzumab,margetuximab, and pertuzumab, and inhibitors of Her2 kinase activity,such as lapatinib, neratinib, and afatinib (which all also inhibitepidermal growth factor receptor (EGFR)). The rexinoid IRX4204 has shownactivity against a variety of cancers in model systems (see for example,U.S. Patent Pub. 2008-0300312, which is incorporated herein by referencein its entirety for all that it teaches about the use of RXR agonistsfor the treatment of cancer) especially when used in combination withthyroid hormone (see for example, U.S. Patent Pub. 2008-0300312, whichis incorporated herein by reference in its entirety for all that itteaches about the use of RXR agonists for the treatment of cancer). Itis disclosed herein that the combination of IRX4204 and a Her2-targetedtherapeutic agent are particularly effective against Her2⁺ cancers, suchas Her2⁺ breast cancer. Thus, various embodiments include combinationsof IRX4204 and a Her2-targeted therapeutic agent, and methods oftreatment involving administration of both IRX4204 and a Her2-targetedtherapeutic agent. Some embodiments further include thyroid hormone orthe administration of thyroid hormone. Other embodiments exclude thyroidhormone or the administration of thyroid hormone.

A Her2-targeted therapeutic agent as used herein is a therapeutic agentthat inhibits growth of cancer cells by inhibiting Her2 function. Suchagents include mAbs that bind to Her2, antibody-drug conjugatescomprising such mAbs, and inhibitors of Her2 tyrosine kinase activity.Collectively, such agents can be referred to as means fortherapeutically targeting Her2. Some embodiments specifically include orare limited to one or more of these classes of agent, or one or morespecies within one or more of these classes of agent. Some embodimentsspecifically exclude one or more of these classes of agent, or one ormore species within one or more of these classes of agent. Thus, meansfor therapeutically targeting Her2 are mAbs that bind to Her2,antibody-drug conjugates comprising mAbs that bind to Her2, andinhibitors of Her2 tyrosine kinase activity. In some embodiments, themAb in an antibody-drug conjugate has therapeutic activity by itself, inother embodiments it does not.

Many embodiments comprise administration of a single anti-Her2 mAb, butsome embodiments comprise administration of multiple anti-Her2 mAbs, forexample, trastuzumab and pertuzumab. While the disclosed embodiments aregenerally described as using anti-Her2 mAbs, further embodiments cansubstitute anti-Her2 polyclonal antiserum for anti-Her2 mAb. Someembodiments can comprise administration of additional mAbs targetingother antigens, some embodiments specifically exclude administration ofother antibodies.

Anti-Her2 Antibodies

Her2 is a growth factor receptor found on, and implicated in, a varietyof cancers, especially breast cancer but also, for example,gastroesophageal cancer, ovarian cancer, stomach cancer, adenocarcinomaof the lung, uterine cancer (such as serous endometrial carcinoma), orsalivary duct carcinoma. Anti-Her2 antibodies are believed to work asanticancer agents through a combination of mechanisms including, but notlimited to, inhibition of signaling through Her2, antibody-dependentcellular cytotoxicity (ADCC), and mediating presentation of tumorantigen by antigen presenting cells (APC), such as macrophages;additional mechanisms may also exist. The best understood and mostclinically advanced anti-Her2 antibodies are trastuzumab, pertuzumab,and margetuximab, although the presently disclosed methods are notlimited to these anti-Her2 mAbs.

In some embodiments, these antibodies are administered by intravenousinfusion. In exemplary embodiments infusions may be done over 30-90minutes and may occur at intervals of 1-3 weeks for as long as a year.In some such embodiments, the antibody is administered at an initialhigher dose and a subsequent lower dose. In some such embodiments, theinitial higher dosage is twice the subsequent lower dosage. In some suchembodiments, the initial dose is single administration. In other suchembodiments the initial dosage is administered multiple times beforeswitching to the lower subsequent dosage. Specific examples of dosagesand dose regimens can be found in the prescribing information forHERCEPTIN® and PERJETA®, which are incorporated herein by reference intheir entireties.

Trastuzumab binds to domain IV of the extracellular segment of Her2.Trastuzumab inhibits the proliferation of cells that overexpress Her2and mediates ADCC. Biosimilar antibodies to trastuzumab have beendeveloped and are marketed in some jurisdictions.

Pertuzumab recognizes the extracellular dimerization domain (domain II)of Her2, a different epitope than trastuzumab. By preventingligand-dependent dimerization, it inhibits signaling through Her2,leading to cell growth arrest and apoptosis. Pertuzumab also mediatesADCC. Pertuzumab augments the activity of trastuzumab in tumor xenograftmodels that over express Her2.

Margetuximab recognizes the same epitope as trastuzumab, but possessesan engineered Fc region designed to increase Fc-dependent mechanisms ofimmune attack, such as ADCC. The engineered Fc region confers increasedbinding to activating Fc-γ receptors (CD16A) and reduced binding toinhibitory Fc-γ receptors (CD16B) on immune effector cells, includingmonocytes, macrophages, dendritic cells and natural killer (NK) cells.

Further anti-Her2 mAbs include TrasGEX®, HM2, hertuzumab, and HT-19.TrasGEX® and HM2 are being developed as “biobetters” of trastuzumab,while the other two are independently derived. In HM2, a metal-bindingmotif has been incorporated into trastuzumab to aid conjugation.TrasGEX® is a glycosylation-optimized version of trastuzumab. Hertuzumabhad a higher ELISA-based affinity for Her2 than trastuzumab. HT-19 is anIgG1 antibody that is non-competitive for HER2 binding with trastuzumaband pertuzumab; that is, it binds a different epitope than either ofthose two mAbs.

Many embodiments comprise administration of a single anti-Her2 mAb, butsome embodiments comprise administration of multiple anti-Her2 mAbs, forexample, trastuzumab and pertuzumab. While the disclosed embodiments aregenerally described as using anti-Her2 mAbs, further embodiments cansubstitute anti-Her2 polyclonal antiserum for anti-Her2 mAb.

Antibody-Drug Conjugates

In addition to use of anti-Her2 mAbs themselves as therapeutic agents,anti-Her2 mAbs have also been incorporated into antibody-drugconjugates. One example is ado-trastuzumab emtansine (KADCYLA®). Furtherexamples include A166, ALT-P7 (trastuzumab biobetter HM2 conjugated in asite-specific manner to monomethyl auristatin E), ARX788 (a monoclonalHER2 targeting antibody site-specifically conjugated, via a non-naturalamino acid linker para-acetyl-phenylalanine (pAcF), to monomethylauristatin F), DHES0815A (a monoclonal HER-2 targeting antibody linkedto pyrrolo[2,1-c][1,4]benzodiazepine monoamide), DS-8201a (trastuzumabderuxtecan; trastuzumab, an enzymatically cleavable maleimideglycynglycyn-phenylalanyn-glycyn (GGFG) peptide linker and atopoisomerase I inhibitor, fam-trastuzumab deruxtecan (ENHERTU®), RC48(humanized anti-HER2 antibody hertuzumab conjugated with monomethylauristatin E (MMAE) via a cleavable linker), SYD985 (([vic-]trastuzumabduocarmazine; trastuzumab linked via a cleavable valine-citrullinepeptide to the synthetic duocarmycin analogseco-duocarmycin-hydroxybenzamide azaindole), MEDI4276 (HER2-bispecificantibody targeting two different epitopes on HER2, site-specificallyconjugated via a maleimidocaproyl linker to the potent tubulysin-basedmicrotubule inhibitor AZ13599185) and XMT-1522 (TAK-522; HT-19conjugated with the DOLAFLEXIN® platform to auristatinF-hydroxypropylamide).

In some embodiments, the Her2-targeting component comprises, or is, anantibody-drug conjugate comprising an anti-Her2 antibody.

Her2 Kinase Inhibitors

Her2 and EGFR are closely related protein tyrosine kinases and manydrugs developed as an inhibitor of one also inhibit the other. At leastfour drugs that are irreversible inhibitors of these kinases are nowmarketed as a cancer treatment (lapatinib, neratinib, afatinib, anddacomitinib), though the indications vary. It should be noted that notall EGFR inhibitors are irreversible inhibitors or are known tocross-inhibit Her2. EGFR inhibitors that do not—or are not knownto—inhibit Her2 should not be considered Her2 inhibitors as the term isused herein. In some embodiments, the Her2 inhibitor is an irreversibleinhibitor. In some embodiments, the Her2 inhibitor is not a reversibleinhibitor. In some embodiments, the Her2 inhibitor is a reversibleinhibitor.

Lapatinib (TYKERB®) (CAS No. 231277-92-2), typically provided asLapatinib Ditosylate (CAS No. 388082-77-7) or the monohydrate thereof,can be administered, according to its prescribing instructions which areincorporated herein by reference in their entirety, on a 21 daytreatment cycle, at 1250 or 1500 mg once daily, depending on indication.Lapatinib plus capecitabine is taken on days 1 to 14. Lapatinib alone istaken on days 15 to 21. At the end of the 21 days, the treatment cycleshould be repeated until disease progression or unacceptable toxicityoccurs. Capecitabine is administered orally in two doses approximately12 hours apart at a dosage of 2000 mg/m²/day.

Neratinib (NERLYNX®) (CAS No. 698387-09-6) can be administered,according to its prescribing instructions which are incorporated hereinby reference in their entirety, with food at an initial dose of 240mg/day and taken daily for a year. If toxicity exceeds grade 1, the dosecan be reduced by 40 mg/day in stepwise fashion until toxicity is grade1 or less. If the dose has been reduced to 120 mg/day and toxicityremains greater than grade 1, treatment with Neratinib should bediscontinued.

Afatinib (GILOTRIF®) (CAS No. 850140-72-6) can be administered,according to its prescribing instructions which are incorporated hereinby reference in their entirety, orally without food once daily at 40mg/day until disease progression or no longer tolerated by the patient.

Dacomitinib (VIZIMPRO®) (CAS No. 1110813-31-4) can be administered,according to its prescribing instructions which are incorporated hereinby reference in their entirety, orally with or without food, once dailyat 45 mg/day until disease progression or unacceptable toxicity occurs.Upon occurrence of unacceptable toxicity, the dosage can be reduced instepwise fashion to 30 or 15 mg/day.

Tucatinib (TUKYSA®) (CAS No. 937263-43-9) can be administered, accordingto its prescribing instructions which are incorporated herein byreference in their entirety, orally with or without food, 300 mg twicedaily (12 hours apart) in combination with trastuzumab and capecitabineuntil disease progression or unacceptable toxicity. Upon occurrence ofunacceptable toxicity, the dosage can be reduced in stepwise fashion, 50mg per step, to 150 mg, twice daily.

The above dosing information, in addition to disclosing specificembodiments in which these Her2 kinase inhibitors may be used incombination with an RXR agonist, provides general guidance as to dosingpractices with such drugs. The disclosed embodiments are not necessarilylimited to these specific dosing regimens and it is within the skill ofthe physician to modify these regimens for individual patients. Due tothe improved and synergistic effect of these drugs when used incombination with a RXR agonist, unacceptable toxicity may be avoided byuse of lower dosages of the kinase inhibitor while still achievebeneficial therapeutic effect.

Other Her2 kinase inhibitors include canertinib (CAS No. 267243-28-7),sapitinib (CAS No. 848942-61-0), CP-724714 (CAS No. 537705-08-1), andCUDC-101 (CAS No. 1012054-59-9).

RXR Agonists

Preclinical studies with rexinoids suggest that selective activation ofRetinoid X Receptors (RXR), which modulate functions associated withdifferentiation, inhibition of cell growth, apoptosis and metastasis,may be useful in treating a variety of diseases associated with thebiochemical functions modulated by RXR.

The Retinoic Acid Receptors (RARs) and RXRs and their cognate ligandsfunction by distinct mechanisms. The term “RAR” as used herein refers toone or more of RARα, RARβ, or RARγ. The term “RXR” as used herein refersto one or more of RXRα, RXRβ, or RXRγ. A RAR biomarker is a distinctivebiological, biochemical or biologically derived indicator that signifiespatient RAR activity. RAR biomarkers include, but are not limited to,CYP26 levels, CRBPI levels, and the like, and combinations thereof.

In some embodiments, the RAR activation threshold means one or more of aCYP26 level which is 25% increased over baseline and a CRBPI level 25%increased over baseline. The RARs form heterodimers with RXRs and theseRAR/RXR heterodimers bind to specific response elements in the promoterregions of target genes. The binding of RAR agonists to the RAR receptorof the heterodimer results in activation of transcription of targetgenes leading to retinoid effects. The disclosed RXR agonists do notactivate RAR/RXR heterodimers. RXR heterodimer complexes like RAR/RXRcan be referred to as non-permissive RXR heterodimers as activation oftranscription due to ligand-binding occurs only at the non-RXR protein(e.g., RAR); activation of transcription does not occur due to ligandbinding at the RXR.

RXRs also interact with nuclear receptors other than RARs and RXRagonists and may elicit some of their biological effects by binding tosuch RXR/receptor complexes. These RXR/receptor complexes can bereferred to as permissive RXR heterodimers as activation oftranscription due to ligand-binding could occur at the RXR, the otherreceptor, or both receptors. Examples of permissive RXR heterodimersinclude, without limitation, peroxisome proliferator activatedreceptor/RXR (PPAR/RXR), farnesyl X receptor/RXR (FXR/RXR), nuclearreceptor related-1 protein (Nurr1/RXR) and liver X receptor/RXR(LXR/RXR). Alternately, RXRs may form RXR/RXR homodimers which can beactivated by RXR agonists leading to rexinoid effects. Also, RXRsinteract with proteins other than nuclear receptors and ligand bindingto an RXR within such protein complexes can also lead to rexinoideffects. Due to these differences in mechanisms of action, RXR agonistsand RAR agonists elicit distinct biological outcomes and even in theinstances where they mediate similar biological effects, they do so bydifferent mechanisms. Moreover, the unwanted side effects of retinoids,such as pro-inflammatory responses or mucocutaneous toxicity, aremediated by activation of one or more of the RAR receptor subtypes.Stated another way, biological effects mediated via RXR pathways wouldnot induce pro-inflammatory responses, and thus, would not result inunwanted side effects.

Thus, aspects of the present specification provide, in part, a RXRagonist. As used herein, the term “RXR agonist”, is synonymous with“selective RXR agonist” and refers to a compound that selectively bindsto one or more RXR receptors like a RXRα, a RXRβ, or a RXRγ in a mannerthat elicits gene transcription via an RXR response element. As usedherein, the term “selectively binds,” when made in reference to a RXRagonist, refers to the discriminatory binding of a RXR agonist to theindicated target receptor like a RXRα, a RXRβ, or a RXRγ such that theRXR agonist does not substantially bind with non-target receptors like aRARα, a RARβ or a RARγ. In some embodiments, the term “RXR agonist”includes esters of RXR agonists.

For each of the herein disclosed embodiments, the RXR agonists can becompounds having the structure of Formula I

wherein R is H, or lower alkyl of 1 to 6 carbons, or a pharmaceuticallyacceptable salt thereof.

Also disclosed herein are esters of RXR agonists. An ester may bederived from a carboxylic acid of Cl, or an ester may be derived from acarboxylic acid functional group on another part of the molecule, suchas on a phenyl ring. While not intending to be limiting, an ester may bean alkyl ester, an aryl ester, or a heteroaryl ester. The term alkyl hasthe meaning generally understood by those skilled in the art and refersto linear, branched, or cyclic alkyl moieties. 01-6 alkyl esters areparticularly useful, where alkyl part of the ester has from 1 to 6carbon atoms and includes, but is not limited to, methyl, ethyl, propyl,isopropyl, n-butyl, sec-butyl, iso-butyl, t-butyl, pentyl isomers, hexylisomers, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, andcombinations thereof having from 1-6 carbon atoms, etc. In someembodiments the RXR agonist is the ethyl ester of formula I.

In some embodiments the RXR agonist is3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydron-aphth-7-yl]2(E),4(E) heptadienoic acid, also known as IRX4204, and has the followingchemical structure:

Pharmaceutically acceptable salts of RXR agonists can also be used inthe disclosed embodiments. Compounds disclosed herein which possess asufficiently acidic, a sufficiently basic, or both functional groups,and accordingly can react with any of a number of organic or inorganicbases, and inorganic and organic acids, to form a salt.

Acids commonly employed to form acid addition salts from RXR agonistswith basic groups are inorganic acids such as hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, andthe like, and organic acids such as p-toluenesulfonic acid,methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonicacid, succinic acid, citric acid, benzoic acid, acetic acid, and thelike. Examples of such salts include the sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenyl butyrate,citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like.

Bases commonly employed to form base addition salts from RXR agonistswith acidic groups include, but are not limited to, hydroxides of alkalimetals such as sodium, potassium, and lithium; hydroxides of alkalineearth metal such as calcium and magnesium; hydroxides of other metals,such as aluminum and zinc; ammonia, and organic amines, such asunsubstituted or hydroxy-substituted mono-, di-, or trialkylamines;dicyclohexylamine; tributyl amine; pyridine; N-methyl,N-ethylamine;diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkylamines), such as mono-, bis-, or tris-(2-hydroxyethyl)amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine,N,N-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)amine, or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike.

IRX4204, like some other RXR ligands, does not activate non-permissiveheterodimers such as RAR/RXR. However, IRX4204, is unique in that itspecifically activates the Nurr1/RXR heterodimer and does not activateother permissive RXR heterodimers such as PPAR/RXR, FXR/RXR, andLXR/RXR. Other RXR ligands generally activate these permissive RXRheterodimers. Thus, all RXR ligands cannot be classified as belonging toone class. IRX4204 belongs to a unique class of RXR ligands whichspecifically activate RXR homodimers and only one of the permissive RXRheterodimers, namely the Nurr1/RXR heterodimer.

In one embodiment, the selective RXR agonist does not activate to anyappreciable degree the permissive heterodimers PPAR/RXR, FXR/RXR, andLXR/RXR. In another embodiment, the selective RXR agonist, activates thepermissive heterodimer Nurr1/RXR. One example of such a selective RXRagonist is3,7-dimethyl-6(S),7(S)-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]2(E),4(E)heptadienoic acid (IRX4204) disclosed herein, the structure of which isshown in Formula II. In other aspects of this embodiment, the RXRagonists activates the permissive heterodimers PPAR/RXR, FXR/RXR, orLXR/RXR by 1% or less, 2% or less, 3% or less, 4% or less, 5% or less,6% or less, 7% or less, 8% or less, 9% or less, or 10% or less relativeto the ability of activating agonists to the non-RXR receptor toactivate the same permissive heterodimer. Examples of RXR agonists,which activates one or more of PPAR/RXR, FXR/RXR, or LXR/RXR includeLGD1069 (bexarotene) and LGD268.

Binding specificity is the ability of a RXR agonist to discriminatebetween a RXR receptor and a receptor that does not contain its bindingsite, such as a RAR receptor.

Particular embodiments provide methods of treating cancer comprisingadministering to a patient in need of such treatment a RXR agonist at alevel below an RAR activating threshold and at or above an RXRactivating threshold.

For IRX4204, the RAR EC₁₀ (the concentration effective to cause a 10% ofmaximal activation of the RAR) is 300 nM for the α isoform and 200 nMfor the β and γ isoforms. Thus, in some embodiments, concentrations notexceeding 200 nM are considered to be below an RAR activatingconcentration. For IRX4204, the RXR EC₉₀ (the concentration effective tocause a 90% of maximal activation of the RXR) is 0.1 nM for the α and γisoforms and 1 nM for the β isoform. Thus, in some embodimentsconcentrations of at least 0.1 nM are considered to be above an RXRactivating threshold. Based on studies in humans, oral dosages ofIRX4204 of 20 mg/m²/day will produce systemic concentrations that remainbelow 200 nM. Similarly, it is estimated that an oral dosage in therange of 0.01 to 0.02 mg/m²/day will produce systemic concentrations of0.1 nM or greater. Thus, in various individual embodiments a dosage ofIRX4204 is at least 0.01, 0.02, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 3 or 5mg/m²/day and does not exceed 150, 200, or 300 mg/m²/day, or any rangebound by a pair of these values.

In other embodiments, the dosage for a human adult of the RXR agonist,for example IRX4204, is from 0.2 to 300 mg/day, such as in individualembodiments, from 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/day, but notto exceed 10, 15, 20, 50, or 100 mg/day, or any range bound by a pair ofthese values.

The RXR agonist can be administered to a mammal using standardadministration techniques, including parenteral, oral, intravenous,intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular,intranasal, buccal, sublingual, or suppository administration. The term“parenteral,” as used herein, includes intravenous, intramuscular,subcutaneous, rectal, vaginal, and intraperitoneal administration. TheRXR agonist preferably is suitable for oral administration, for exampleas a pill, tablet or capsule. Administration may be continuous orintermittent. In certain embodiments, the total daily dosage of RXRagonist can be administered as a single dose or as two dosesadministered with a 24 hour period spaced 8 to 16, or 10 to 14, hoursapart.

Thyroid Hormone

Both biologically sourced and synthetic thyroid hormones have been usedin medicine. The major forms of thyroid hormone are referred to as T₃(triiodothyronine) and T₄ (thyroxine). Thyroxine is less active but hasa longer half-life and is sometimes considered a prohormone oftriiodothyronine. As used herein, the term “thyroid hormone” refers toboth thyroxine and triiodothyronine. Thyroxine (thyroid hormone T₄,levothyroxine sodium) is a tyrosine-based hormone produced by thethyroid gland and is primarily responsible for regulation of metabolism.Both have wide commercial availability and are suitable for use in theherein disclosed embodiments. However, the synthetic form of T₄,levothyroxine, is much more commonly utilized clinically (except inpatients unable to convert T₄ into T₃) as its longer half-life in thebody facilitates once-daily administration. In some embodiments, theadministered thyroid hormone is specifically thyroxine. In someembodiments, the administered thyroid hormone is triiodothyronine.

Administration of RXR agonists, or esters thereof, may lead to thesuppression of serum thyroid hormones and possibly to clinicalhypothyroidism and related conditions. However, in some embodimentsthyroid hormone is not co-administered (or is not primarilyco-administered) to remediate a suppression of serum thyroid hormonelevels. Co-administration of thyroid hormone with an RXR agonistimproves the RXR agonist's anti-cancer efficacy, as compared to theeffect of the RXR agonist alone, likely through multiple mechanisms ofaction. The co-administered thyroid hormone can also mitigate thehypothyroid-inducing effects of the RXR agonist, thereby improving theclinical safety and tolerability of the treatment. Thus, in preferredembodiments, thyroid hormone is co-administered with an RXR agonist toimprove the efficacy of the treatment, whether or not administration ofthe RXR agonist has caused, or is expected to cause, clinicalhypothyroidism. By administration of thyroid hormone in coordination orin conjunction with the RXR agonist it is meant that the manner ofadministration of each of these two agents is such that the physiologiceffects of the two agents overlap in time. This does not require thatthe RXR agonist and thyroid hormone be contained in the same compositionor formulation, or that they be administered as separate compositions atthe same time, by the same route of administration, or on the sameschedule, though in some embodiments any of the foregoing may be thecase.

Suitable thyroxine doses are generally from about 5 μg/day to about 250μg/day orally initially with an increase in dose every 2-4 weeks asneeded. In other embodiments, the suitable thyroxine dose is from about5 μg/day to about 225 μg/day, from about 7.5 μg/day to about 200 μg/day,from about 10 μg/day to about 175 μg/day, from about 12.5 μg/day toabout 150 μg/day, from about 15 μg/day to about 125 μg/day, from about17.5 μg/day to about 100 μg/day, from about 20 μg/day to about 100μg/day, from about 22.5 μg/day to about 100 μg/day, from about 25 μg/dayto about 100 μg/day, from about 5 μg/day to about 200 μg/day, from about5 μg/day to about 100 μg/day, from about 7.5 μg/day to about 90 μg/day,from about 10 μg/day to about 80 μg/day, from about 12.5 μg/day to about60 μg/day, or from about 15 μg/day to about 50 μg/day. Increases in doseare generally made in increments of about 5 μg/day, about 7.5 μg/day,about 10 μg/day, about 12.5 μg/day, about 15 μg/day, about 20 μg/day, orabout 25 μg/day. In certain embodiments, the suitable thyroid hormonedose is a dose able to produce serum levels of T₄ in the top 50%, thetop 60%, the top 70%, the top 80%, or the top 90% of the normal rangefor the testing laboratory. As the normal range of T₄ levels may vary bytesting laboratory, the target T₄ levels are based on normal rangesdetermined for each particular testing laboratory.

For each embodiment involving a combination of RXR agonist andHer2-targeting therapeutic agent, there is a parallel embodiment inwhich the combination further comprises thyroid hormone.

Pharmaceutical Compositions and Formulations

The various component active agents used in the herein describedtreatments will typically exist as pharmaceutical compositions orformulations. Such compositions or formulations may be a liquidformulation, semi-solid formulation, or a solid formulation. Aformulation disclosed herein can be produced in a manner to form onephase, such as, e.g., an oil or a solid. Alternatively, a formulationdisclosed herein can be produced in a manner to form two phases, suchas, e.g., an emulsion. A pharmaceutical composition disclosed hereinintended for such administration may be prepared according to any methodknown to the art for the manufacture of pharmaceutical compositions.

Liquid formulations suitable for parenteral injection or for nasalsprays may comprise physiologically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions and sterilepowders for reconstitution into sterile injectable solutions ordispersions. Formulations suitable for nasal administration may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (propylene glycol, polyethyleneglycol (PEG), glycerol,and the like), suitable mixtures thereof, vegetable oils (such as oliveoil) and injectable organic esters such as ethyl oleate. Proper fluiditycan be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions and by the use of surfactants.

A pharmaceutical composition disclosed herein can optionally include apharmaceutically acceptable carrier that facilitates processing of anactive compound into pharmaceutically acceptable compositions. As usedherein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem complicationscommensurate with a reasonable benefit/risk ratio. This definition isalso application to the phrase “pharmaceutically-acceptable salts”. Asused herein, the term “pharmacologically acceptable carrier” issynonymous with “pharmacological carrier” and refers to any carrier thathas substantially no long term or permanent detrimental effect whenadministered and encompasses terms such as “pharmacologically acceptablevehicle, stabilizer, diluent, additive, auxiliary, or excipient.” Such acarrier generally is mixed with an active compound or permitted todilute or enclose the active compound and can be a solid, semi-solid, orliquid agent. It is understood that the active compounds can be solubleor can be delivered as a suspension in the desired carrier or diluent.Any of a variety of pharmaceutically acceptable carriers can be usedincluding, without limitation, aqueous media such as, e.g., water,saline, glycine, hyaluronic acid and the like; solid carriers such as,e.g., starch, magnesium stearate, mannitol, sodium saccharin, talcum,cellulose, glucose, sucrose, lactose, trehalose, magnesium carbonate,and the like; solvents; dispersion media; coatings; antibacterial andantifungal agents; isotonic and absorption delaying agents; or any otherinactive ingredient. Selection of a pharmacologically acceptable carriercan depend on the mode of administration. Except insofar as anypharmacologically acceptable carrier is incompatible with the activecompound, its use in pharmaceutically acceptable compositions iscontemplated. Non-limiting examples of specific uses of suchpharmaceutical carriers can be found in Pharmaceutical Dosage Forms andDrug Delivery Systems (Howard C. Ansel et al., eds., Lippincott Williams& Wilkins Publishers, 7^(th) ed. 1999); Remington: The Science andPractice of Pharmacy (Alfonso R. Gennaro ed., Lippincott, Williams &Wilkins, 20^(th) ed. 2000); Goodman & Gilman's The Pharmacological Basisof Therapeutics (Joel G. Hardman et al., eds., McGraw-Hill Professional,10^(th) ed. 2001); and Handbook of Pharmaceutical Excipients (Raymond C.Rowe et al., APhA Publications, 4th edition 2003). These protocols areroutine and any modifications are well within the scope of one skilledin the art and from the teaching herein.

A pharmaceutical composition disclosed herein can optionally include,without limitation, other pharmaceutically acceptable components (orpharmaceutical components), including, without limitation, buffers,preservatives, tonicity adjusters, salts, antioxidants, osmolalityadjusting agents, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed herein,provided that the resulting preparation is pharmaceutically acceptable.Such buffers include, without limitation, acetate buffers, boratebuffers, citrate buffers, phosphate buffers, neutral buffered saline,and phosphate buffered saline. It is understood that acids or bases canbe used to adjust the pH of a composition as needed. Pharmaceuticallyacceptable antioxidants include, without limitation, sodiummetabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole, and butylated hydroxytoluene. Useful preservativesinclude, without limitation, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, phenylmercuric nitrate, a stabilizedoxy chloro composition, such as, e.g., sodium chlorite and chelants,such as, e.g., DTPA or DTPA-bisamide, calcium DTPA, andCaNaDTPA-bisamide. Tonicity adjustors useful in a pharmaceuticalcomposition include, without limitation, salts such as, e.g., sodiumchloride, potassium chloride, mannitol or glycerin and otherpharmaceutically acceptable tonicity adjustor. The pharmaceuticalcomposition may be provided as a salt and can be formed with many acids,including but not limited to, hydrochloric, sulfuric, acetic, lactic,tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueousor other protonic solvents than are the corresponding free base forms.It is understood that these and other substances known in the art ofpharmacology can be included in a pharmaceutical composition useful inthe invention.

Pharmaceutical formulations suitable for administration by inhalationinclude fine particle dusts or mists, which may be generated by means ofvarious types of metered, dose pressurized aerosols, nebulizers, orinsufflators.

Semi-solid formulations suitable for topical administration include,without limitation, ointments, creams, salves, and gels. In such solidformulations, the active compound may be admixed with at least one inertcustomary excipient (or carrier) such as, a lipid and/or polyethyleneglycol.

Solid formulations suitable for oral administration include capsules,tablets, pills, powders and granules. In such solid formulations, theactive compound may be admixed with at least one inert customaryexcipient (or carrier) such as sodium citrate or dicalcium phosphate or(a) fillers or extenders, as for example, starches, lactose, sucrose,glucose, mannitol and silicic acid, (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate or mixturesthereof. In the case of capsules, tablets and pills, the dosage formsmay also comprise buffering agents.

The small molecule components of the various embodiments, that is, theRXR agonist, thyroid hormone, and Her2 kinase inhibitors are capable ofbeing formulated in solid, oral dosage forms. The antibody componentsare generally formulated as liquids typically for intravenous infusion.In alternative embodiments the antibody components may be supplied inlyophilized form for reconstitution as a liquid locally at the site oftreatment, where they are also typically infused intravenously into thepatient. While intravenous infusion is typical, in alternativeembodiments the antibody may be administered by another route ofadministration, such as subcutaneous injection or infusion.

Treatment

As used herein, the terms “treatment,” “treating,” and the like refer toobtaining a desired pharmacologic and/or physiologic effect. This may beobserved directly as a slowing of tumor growth, stabilization ofdisease, or a partial or complete response (that is, tumor regression orelimination of tumors), or extended overall or disease-free survival.Treatment may also be observed as an amelioration or reduction ofsymptoms related to the underlying cancer. However, as cancer treatment,the disclosed embodiments' aim and mechanism is directed to inhibiting,stabilizing, or reducing tumor growth (including metastases), orpartially or completely eliminating tumors, or extending overall ordisease-free survival; effects on other cancer symptoms are secondary.Direct treatment of such other symptoms (for example, pain, nausea, lossof appetite, etc.) is not within the scope of treating cancer as usedherein. That is, treating a symptom, for example, cachexia in a cancerpatient is not treating cancer. However, an agent that treats cancer(e.g., has an impact on the growth and/or spread of cancer) may alsoameliorate a symptom, such as cachexia, either indirectly, through itseffect on the cancer, or directly, through a pleiotropic effect. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve a desiredtherapeutic result. The therapeutically effective amount may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the Her2-targeted therapy, RXRagonist, and if used, thyroid hormone, to elicit a desired response inthe individual.

However, the dose administered to a mammal, particularly a human, in thecontext of the present methods, should be sufficient to effect atherapeutic response in the mammal over a reasonable timeframe. Oneskilled in the art will recognize that the selection of the exact doseand composition and the most appropriate delivery regimen will also beinfluenced by inter alia the pharmacological properties of theformulation, the nature and severity of the condition being treated, andthe physical condition and mental acuity of the recipient, as well asthe potency of the specific compound, the age, condition, body weight,sex and response of the patient to be treated, and the stage/severity ofthe disease.

Treatment activity includes the administration of the medicaments,dosage forms, and pharmaceutical compositions described herein to apatient, especially according to the various methods of treatmentdisclosed herein, whether by a healthcare professional, the patienthis/herself, or any other person. Treatment activities include theorders, instructions, and advice of healthcare professionals such asphysicians, physician's assistants, nurse practitioners, and the likethat are then acted upon by any other person including other healthcareprofessionals or the patient his/herself. In some embodiments, treatmentactivity can also include encouraging, inducing, or mandating that aparticular medicament, or combination thereof, be chosen for treatmentof a condition—and the medicament is actually used—by approvinginsurance coverage for the medicament, denying coverage for analternative medicament, including the medicament on, or excluding analternative medicament, from a drug formulary, or offering a financialincentive to use the medicament, as might be done by an insurancecompany or a pharmacy benefits management company, and the like. In someembodiments, treatment activity can also include encouraging, inducing,or mandating that a particular medicament be chosen for treatment of acondition—and the medicament is actually used—by a policy or practicestandard as might be established by a hospital, clinic, healthmaintenance organization, medical practice or physicians group, and thelike.

To benefit from the combined effect of a RXR agonist of Formula I (orpharmaceutically acceptable salt or ester thereof) and Her2-targetedtherapeutics, embodiments include methods of treatment comprising orconsisting of administering RXR agonist of Formula I (orpharmaceutically acceptable salt thereof) and a Her2-targetedtherapeutic to a patient having a Her2⁺ cancer. Some embodiments furthercomprise administration of thyroid hormone in coordination withadministration of the RXR agonist. In some embodiments the Her2⁺ canceris a Her2⁺ breast cancer. In some embodiments the Her2⁺ cancer is aHer2⁺ gastroesophageal cancer, ovarian cancer, stomach cancer,adenocarcinoma of the lung, uterine cancer (such as serous endometrialcarcinoma), or salivary duct carcinoma. Some embodiments specificallyinclude one or more of these cancers. Other embodiments specificallyexclude one or more of these cancers.

In various embodiments the herein disclosed treatments may be applied asa primary therapy, as a debulking therapy prior to surgical removal oftumor, or as an adjuvant therapy subsequent to any mode of primarytherapy (especially surgery) to address residual disease and/or lowerthe risk of recurrent cancer.

In some embodiments, the patient having a Her2⁺ cancer has not beenpreviously treated with either RXR agonist of Formula I (orpharmaceutically acceptable salt thereof) or a Her2-targetedtherapeutic. In some embodiments the patient has been previously treatedwith RXR agonist of Formula I (or pharmaceutically acceptable saltthereof) and has achieved stable disease or a partial response (in someembodiments, as defined by RECIST or iRECIST criteria)—that is, thecancer is sensitive to RXR agonist of Formula I (or pharmaceuticallyacceptable salt thereof)—and a Her2-targeted therapeutic is added to thetreatment regimen. In some embodiments the patient has been previouslytreated with a Her2-targeted therapeutic and has achieved stable diseaseor a partial response (in some embodiments, as defined by RECIST oriRECIST criteria)—that is, the cancer is sensitive to a Her2-targetedtherapeutic—and RXR agonist of Formula I (or pharmaceutically acceptablesalt thereof) is added to the treatment regimen.

Thus, some embodiments comprise administration of an RXR agonist to apatient with a Her2⁺ tumor who has received, is receiving, or isscheduled to receive, a Her2-targeted therapeutic agent. Someembodiments comprise administration of an RXR agonist to a patient inwhom a Her2-targeted therapeutic agent has had some therapeutic effect(less than a complete response), that is administration of the RXRagonist is added to the therapeutic regimen for the Her2-targetedtherapeutic agent. Some embodiments comprise administration of aHer2-targeted therapeutic agent to a patient in whom an RXR agonist (orRXR agonist in conjunction with thyroid hormone) has had sometherapeutic effect (less than a complete response), that isadministration of the Her2-targeted therapeutic agent is added to thetherapeutic regimen for the RXR agonist.

Therapeutic efficacy can be monitored by periodic assessment of treatedpatients. For repeated administrations over several days or longer, thetreatment can be repeated until a desired suppression of disease ordisease symptoms occurs. However, other dosage regimens may be usefuland are within the scope of the present disclosure. Antibodies typicallyhave a much longer half-life in the body than the other active agentsused in these methods and therefore there will typically besubstantially longer intervals (measured in weeks) betweenadministrations.

The effectiveness of cancer therapy is typically measured in terms of“response.” The techniques to monitor responses can be similar to thetests used to diagnose cancer such as, but not limited to:

-   -   A lump or tumor involving some lymph nodes can be felt and        measured externally by physical examination.    -   Some internal cancer tumors will show up on an x-ray or CT scan        and can be measured with a ruler.    -   Blood tests, including those that measure organ function can be        performed.    -   A tumor marker test can be done for certain cancers.

Regardless of the test used, whether blood test, cell count, or tumormarker test, it is repeated at specific intervals so that the resultscan be compared to earlier tests of the same type.

Response to cancer treatment is defined several ways:

-   -   Complete response—all of the cancer or tumor disappears; there        is no evidence of disease. Expression level of tumor marker (if        applicable) may fall within the normal range.    -   Partial response—the cancer has shrunk by a percentage but        disease remains. Levels of a tumor marker (if applicable) may        have fallen (or increased, based on the tumor marker, as an        indication of decreased tumor burden) but evidence of disease        remains.    -   Stable disease—the cancer has neither grown nor shrunk; the        amount of disease has not changed. A tumor marker (if        applicable) has not changed significantly.    -   Disease progression—the cancer has grown; there is more disease        now than before treatment. A tumor marker test (if applicable)        shows that a tumor marker has risen.

Other measures of the efficacy of cancer treatment include intervals ofoverall survival (that is time to death from any cause, measured fromdiagnosis or from initiation of the treatment being evaluated)),cancer-free survival (that is, the length of time after a completeresponse cancer remains undetectable), and progression-free survival(that is, the length of time after disease stabilization or partialresponse that resumed tumor growth is not detectable).

There are two standard methods for the evaluation of solid cancertreatment response with regard to tumor size (tumor burden), the WHO andRECIST standards. These methods measure a solid tumor to compare acurrent tumor with past measurements or to compare changes with futuremeasurements and to make changes in a treatment regimen. In the WHOmethod, the solid tumor's long and short axes are measured with theproduct of these two measurements is then calculated; if there aremultiple solid tumors, the sum of all the products is calculated. In theRECIST method, only the long axis is measured. If there are multiplesolid tumors, the sum of all the long axes measurements is calculated.However, with lymph nodes, the short axis is measured instead of thelong axis. There is also a variation of the RECIST method forimmunotherapies (iRECIST) which takes into account distinctive behaviorslinked to these types of therapeutics, such as delayed responses afterpseudoprogression. Both the RECIST 1.1 guidelines and the iRecistguidelines are incorporated by reference herein in their entirety.

In some embodiments of the herein disclosed methods, the tumor burden ofa treated patient is reduced by about 5%, about 10%, about 15%, about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55% about 60%, about 65%, about 70%, about 75%, about 80%, about90%, about 95%, about 100%, or any range bound by these values.

In other embodiments, the 1-year survival rate of treated subjects isincreased by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55% about 60%,about 65%, about 70%, about 75%, about 80%, about 90%, about 95%, about100%, or any range bound by these values.

In other embodiments, the 5-year survival rate of treated subjects isincreased by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55% about 60%,about 65%, about 70%, about 75%, about 80%, about 90%, about 95%, about100%, or any range bound by these values.

In other embodiments, the 10-year survival rate of treated subjects isincreased by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55% about 60%,about 65%, about 70%, about 75%, about 80%, about 90%, about 95%, about100%, or any range bound by these values.

In yet other embodiments, the subject has a sustained remission of atleast 6 months, at least 7 months, at least 8 months, at least 9 months,at least 10 months, at least 11 months, at least 12 months, at least 14months, at least 16 months, at least 18 months, at least 20 months, atleast 22 months, at least 24 months, at least 27 months, at least 30months, at least 33 months, at least 36 months, at least 42 months, atleast 48 months, at least 54 months, or at least 60 months or more.

In other embodiments, the methods may additionally help to treat oralleviate conditions, symptoms, or disorders related to cancer. In someembodiments, these conditions or symptoms may include, but are notlimited to, anemia, asthenia, cachexia, Cushing's syndrome, fatigue,gout, gum disease, hematuria, hypercalcemia, hypothyroidism, internalbleeding, hair loss, mesothelioma, nausea, night sweats, neutropenia,paraneoplastic syndromes, pleuritis, polymyalgia rheumatica,rhabdomyolysis, stress, swollen lymph nodes, thrombocytopenia, vitamin Ddeficiency, or weight loss. While a cancer treatment may reduce or treatassociated symptoms, treating symptoms associated with cancer, is nottreating cancer if there is no expectation that tumor will be reduced oreliminated or their growth or spread will be inhibited.

Toxicities and adverse events are sometimes graded according to a 5point scale. A grade 1 or mild toxicity is asymptomatic or induces onlymild symptoms; may be characterized by clinical or diagnosticobservations only; and intervention is not indicated. A grade 2 ormoderate toxicity may impair activities of daily living (such aspreparing meals, shopping, managing money, using the telephone, etc.)but only minimal, local, or non-invasive interventions are indicated.Grade 3 toxicities are medically significant but not immediatelylife-threatening; hospitalization or prolongation of hospitalization isindicated; activities of daily living related to self-care (such asbathing, dressing and undressing, feeding oneself, using the toilet,taking medications, and not being bedridden) may be impaired. Grade 4toxicities are life-threatening and urgent intervention is indicated.Grade 5 toxicity produces an adverse event-related death. Thus invarious embodiments, use of an RXR agonist, or RXR agonist and thyroidhormone, reduces the grade of a toxicity that would otherwise beassociated with use of the Her2-targeted therapy, by allowing a lowerdose to be used without substantial sacrifice of efficacy. In someembodiments, use of an RXR agonist, or RXR agonist and thyroid hormone,in combination with the Her2-targeted therapeutic agent limits atoxicity to grade 1 or less, or produces no observation of the toxicity,without substantial reduction of efficacy as would be expected from theHer2-targeted therapeutic agent alone. In some embodiments, the combineduse of the Her2-targeted therapeutic agent and the RXR agonist, or RXRagonist and thyroid hormone, allows continued use of the Her2-targetedtherapeutic agent at a lower dosage with therapeutic effect in instanceswhere treatment with the Her2-targeted therapeutic agent would have hadto have been discontinued due to unacceptable toxicity. In some of theseembodiments, the Her2-targeted therapeutic agent comprises a Her2 kinaseinhibitor.

The combination of the disclosed RXR agonists and the Her2 targetedtherapeutics are synergistic in effect. That is, they interact in apositive manner to produce a greater inhibition of tumor cell growththan would be expected from the independent (non-interacting) effects ofthe two. Thus, some embodiments produce improved efficacy. Otherembodiments allow for the reduction of dosage in order to reducetoxicity while still achieving at least similar efficacy as provided byan individual therapeutic agent. In some embodiments, both reducedtoxicity and improved efficacy (as compared to the more toxic singleagent) is achieved.

For each method of treatment there are further parallel embodimentsrelated to the foregoing methods directed to use of the RXR agonist inconjunction with a Her2-targeted therapeutic agent, or a Her2-targetedtherapeutic agent and thyroid hormone, to treat Her2⁺ cancer; directedto use of the RXR agonist in the manufacture of a medicament for use incombination with a Her2-targeted therapeutic agent, or a Her2-targetedtherapeutic agent and thyroid hormone, to treat Her2⁺ cancer.

Further embodiments include a combination comprising a RXR agonist asherein described and a Her2-targeted therapeutic agent. Some embodimentsfurther comprise a thyroid hormone. In some embodiments, theHer2-targeted therapeutic agent is an anti-Her2 antibody. In someembodiments the Her2-targeted therapeutic agent is a Her2 kinaseinhibitor.

Further embodiments include kits comprising the above combinations. Thekits may additionally comprise solvents, diluents, injectors, and thelike that may facilitate administration of one or more of thetherapeutic agents. The kits may further comprise instructions for thecoordinated use of the therapeutic agents utilized in the disclosedmethods, whether or not any particular agent is supplied in the kit.

LIST OF PARTICULAR EMBODIMENTS

The following listing of embodiments is illustrative of the variety ofembodiments with respect to breadth, combinations and sub-combinations,class of invention, etc., elucidated herein, but is not intended to bean exhaustive enumeration of all embodiments finding support herein.

Embodiment 1. A method of treating a patient with Her2⁺ cancer resistantto a Her2-targeted therapeutic agent comprising administering a RXRagonist of Formula I,

-   -   wherein R is H, or lower alkyl of 1 to 6 carbons; or a        pharmaceutically-acceptable salt thereof, to the patient,        wherein the patient has received, is receiving, or is scheduled        to receive the Her2-targeted therapeutic agent.

Embodiment 2. A method of treating a patient with Her2+ cancer resistantto a Her2-targeted therapeutic agent comprising administering a RXRagonist of Formula I,

-   -   wherein R is H, or lower alkyl of 1 to 6 carbons; or a        pharmaceutically-acceptable salt thereof, and the Her2-targeted        therapeutic agent.

Embodiment 3. A method of treating a patient with Her2+ cancerundergoing treatment with a RXR agonist of Formula I,

-   -   wherein R is H, or lower alkyl of 1 to 6 carbons; or a        pharmaceutically-acceptable salt thereof,    -   wherein the cancer has become resistant to the RXR agonist,        comprising continuing treatment with the RXR agonist and        initiating treatment with a Her2-targeted therapeutic agent.

Embodiment 4. A method of treating a patient with Her2+ cancerundergoing treatment with a Her2-targeted therapeutic agent, wherein thecancer has become resistant to the Her2-targeted therapeutic agent,comprising continuing treatment with the Her2-targeted therapeutic agentand initiating treatment with a RXR agonist of Formula I,

-   -   wherein R is H, or lower alkyl of 1 to 6 carbons; or a        pharmaceutically-acceptable salt thereof.

Embodiment 5. A method of treating a patient with Her2⁺ cancer resistantto a Her2-targeted therapeutic agent comprising administering a RXRagonist of Formula I,

-   -   wherein R is H, or lower alkyl of 1 to 6 carbons; or a        pharmaceutically-acceptable salt thereof, to the patient,    -   wherein the patient has received, is receiving, or is scheduled        to receive means for therapeutically targeting Her2.

Embodiment 6. The method of Embodiment 5, wherein the means fortherapeutically targeting Her2 are:

-   -   means for inhibiting Her2⁺ tumor cell proliferation,    -   means for mediating ADCC of Her2⁺ tumor cells,    -   immunoglobulin means for inhibiting Her2 signaling,    -   means for delivering a cytotoxic agent to Her2⁺ cells, or    -   small molecule means for inhibiting Her2 kinase activity.

Embodiment 7. A method of treating a patient with Her2⁺ cancer resistantto a Her2-targeted therapeutic agent comprising administering means foractivating RXR/Nurr1 heterodimeric receptors or rexinoid means forinhibiting tumor growth, wherein the patient has received, is receiving,is scheduled to receive a Her2-targeted therapeutic agent.

Embodiment 8. The method of any one of Embodiments 1-7, furthercomprising administering thyroid hormone in conjunction with the RXRagonist.

Embodiment 9. The method of Embodiment 8, wherein the thyroid hormone isthyroxine.

Embodiment 10. The method of any one of Embodiments 1-9, wherein the RXRagonist, the means for activating RXR/Nurr1 heterodimeric receptors, orthe rexinoid means for inhibiting tumor growth, is a compound of FormulaI.

Embodiment 11. The method of any one of Embodiments 1-9, wherein the RXRagonist, the means for activating RXR/Nurr1 heterodimeric receptors, orthe rexinoid means for inhibiting tumor growth, is apharmaceutically-acceptable salt of a compound of Formula I.

Embodiment 12. The method of any one of Embodiments 1-9, wherein the RXRagonist, the means for activating RXR/Nurr1 heterodimeric receptors, orthe rexinoid means for inhibiting tumor growth, is a compound of FormulaII

Embodiment 13. The method of any one of Embodiments 1-12, wherein:

-   -   the Her2-targeted therapeutic agent,    -   the means for inhibiting Her2⁺ tumor cell proliferation,    -   the means for mediating ADCC of Her2⁺ tumor cells,    -   the immunoglobulin means for inhibiting Her2 signaling, or    -   the means for delivering a cytotoxic agent to Her2⁺ cells,    -   comprises an anti-Her2 therapeutic antibody.

Embodiment 14. The method of Embodiment 13, wherein the therapeuticantibody is trastuzumab or pertuzumab.

Embodiment 15. The method of Embodiment 13, wherein the therapeuticantibody is margetuximab, TrasGEX, HM2, hertuzumab, or HT-19

Embodiment 16. The method of any one of Embodiments 1-13, wherein theHer2-targeted therapeutic agent, the means for inhibiting Her2⁺ tumorcell proliferation, or the immunoglobulin means for inhibiting Her2signaling, comprises an antibody-drug conjugate wherein the antibody isan anti-Her2 antibody.

Embodiment 17. The method of Embodiment 16, wherein the antibody-drugconjugate or the means for delivering a cytotoxic agent to Her2⁺ cells,is ado-trastuzumab emtansine.

Embodiment 18. The method of Embodiment 16, wherein the antibody-drugconjugate or the means for delivering a cytotoxic agent to Her2⁺ cells,is A166, ALT-P7, ARX788, DHES0815A, DS-8201a, RC48, SYD985, MEDI4276, orXMT-1522.

Embodiment 19. The method of any one of Embodiments 1-12, wherein theHer2-targeted therapeutic agent, or the small molecule means forinhibiting Her2 kinase activity, comprises a Her2 kinase inhibitor.

Embodiment 20. The method of Embodiment 19, wherein the Her2 kinaseinhibitor is lapatinib or neratinib.

Embodiment 21. The method of Embodiment 19, wherein the Her2 kinaseinhibitor is afatinib or dacomitinib.

Embodiment 22. The method of any one of Embodiments 1-21, wherein thetreatment is applied as a debulking therapy.

Embodiment 23. The method of any one of Embodiments 1-21, wherein thetreatment is applied as adjuvant therapy.

Embodiment 24. The method of any one of Embodiments 1-23, wherein theHer2⁺ cancer is Her2⁺ breast cancer.

Embodiment 25. The method of any one of Embodiments 1-23, wherein theHer2⁺ cancer is Her2⁺ gastroesophageal cancer, ovarian cancer, stomachcancer, adenocarcinoma of the lung, uterine cancer (such as serousendometrial carcinoma), or salivary duct carcinoma.

Embodiment 26. The method of any one of Embodiments 1-25, wherein atherapeutic response to the RXR agonist and the Her2-targetedtherapeutic agent is greater than to the response to either of theagents alone.

Embodiment 27. The method of Embodiment 26, wherein the greatertherapeutic response is a slowing of tumor growth, stabilization ofdisease, a partial response, a complete response, extended overallsurvival, or disease-free survival.

Embodiment 28. The method of Embodiment 26 or 27, wherein response isevaluated according to RECIST or iRECIST criteria.

Embodiment 29. The method of any one of Embodiments 26-28, comprising areduction or amelioration of secondary symptoms.

Embodiment 30. The method of any one of Embodiments 26-29, wherein theHer2⁺ cancer resistant to a Her2-targeted therapeutic agent is alsoresistant to the RXR agonist.

Embodiment 31. A RXR agonist of Formula I,

-   -   wherein R is H, or lower alkyl of 1 to 6 carbons; or a        pharmaceutically-acceptable salt thereof, for use in        manufacturing a medicament for treating Her2⁺ cancer resistant        to a Her2-targeted therapeutic agent, in a patient who has        received, is receiving, or is scheduled to receive a        Her2-targeted therapeutic agent.

Embodiment 32. A RXR agonist of Formula I,

-   -   wherein R is H, or lower alkyl of 1 to 6 carbons; or a        pharmaceutically-acceptable salt thereof, and a Her2-targeted        therapeutic agent, for use in manufacturing medicaments for use        together to treat Her2⁺ cancer resistant to a Her2-targeted        therapeutic agent.

Embodiment 33. A RXR agonist of Formula I,

-   -   wherein R is H, or lower alkyl of 1 to 6 carbons; or a        pharmaceutically-acceptable salt thereof, for treating Her2⁺        cancer resistant to a Her2-targeted therapeutic agent in a        patient with Her2⁺ cancer, in combination with the Her2-targeted        therapeutic agent.

It should be manifest that each or Embodiments 31-33 can be modified ina manner similar to the modification of Embodiments 1-5 and 7 byEmbodiments 5 and 8-29.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification.

Example 1 Inhibition of Breast Cancer Cell Growth by IRX4204 PlusTrastuzumab

Two breast cancer cell lines, MCF7 and SkBr3, were cultured in thepresence of 0, 10, 100, or 1000 nM IRX4204 and 0, 0.1, 1, or 10 μg/mltrastuzumab. MCF7 is an ER⁺ PR⁺ Her2⁻ cell line. SkBr3 is an ER⁻ PR⁻Her2⁺ cell line. Cells were plated in 96 well optical plates andIRX-4204 and trastuzumab were added 24 hours after cell plating. After afurther 6 days, the cells were fixed with 4% paraformaldehyde inphosphate buffered saline (PBS). Nuclei were then stained with DAPI andimaged with the MetaXpress® microscope (Molecular Devices, San Jose,Calif.). Cell nuclei were segmented and then counted by defining pixelintensity over background and object size, using the algorithm of theMetaXpress® image analysis software. Experimental data points wereperformed at a minimum of quadruplicate, and results were reported asaverage cell count±standard deviation (SD). As seen in FIG. 1A, neitheragent had more than marginal effect on the Her2⁻ MCF7 cell line. As seenin FIG. 1B, both agents individually have a moderate growth inhibitoryeffect on the Her2⁺ SkBr3 cell line, and had a very substantial, growthinhibitory effect when used in combination, even at the lowestconcentrations tested.

To assess if this improved growth inhibition effect was synergistic, thepercent inhibition observed for the highest concentrations of thecombined therapeutic agents was compared to the percent inhibition thatwould be expected for the combination based on the observed inhibitionof the therapeutic agents used alone, if the agents actingindependently, that is, without interaction (see Table 1). That is,PAE=(FE1+((1−FE1)×FE2))×100, where PAE is the predicted additive effect,FE1 is the observed fractional effect of a 1st treatment, and FE2 is theobserved fractional effect of a 2nd treatment.

TABLE 1 Synergistic Effect of the Combination of IRX4204 and Trastuzumabon the Inhibition of the Growth of SkBr3 Cells. DAPI Nuclei TreatmentCount % Inhibition Control 25958 IRX4204 1000 nM 18274 29.4 If Additive= 68.5% Herceptin 1000 ng 11582 55.4 IRX4204 + Herceptin 2946 88.7

Whereas the combined inhibitory effect of IRX4204 and trastuzumab wouldbe predicted to be 68.5% if there was no interaction between the effectsof the two agents, in fact the observed degree of inhibition was 88.7%,clearly indicating that IRX4204 and trastuzumab interact in asynergistic manner.

Example 2 Inhibition of Breast Cancer Cell Growth by IRX4204 PlusLapatinib or Neratinib

The experiment of Example 1 was repeated using the Her2 kinaseinhibitors lapatinib or neratinib instead of trastuzumab at 0, 0.1, 1,or 10 nM. Additionally the panel of breast cancer cell lines wasexpanded to also include the ER⁺ PR⁺ Her2⁺ cell line BT474 and the ER⁺PR⁻ Her2⁺ cell line MDA-MB-361. The general pattern seen above, that theHer2⁻ MCF7 cell line showed generally marginal response to thetreatments and that the Her⁺ cell lines exhibited a greater degree ofinhibition to the combination than either agent alone, was againobserved (see FIGS. 2 and 3 ).

To assess if this improved growth inhibition effect was synergistic, thepercent inhibition observed for the highest concentrations of thecombined therapeutic agents was compared to the percent inhibition thatwould be expected for the combination based on the observed inhibitionof the therapeutic agents used alone, if the agents actingindependently, that is, without interaction (see Tables 2-4).

TABLE 2 Synergistic Effect of the Combination of IRX4204 and Lapatinibor Neratinib on the Inhibition of the Growth of SkBr3 Cells. DAPI NucleiTreatment Count % Inhibition Control 19855 IRX4204 1000 nM 18906 4.8 IfAdditive = 89.4% Lapatinib 1000 nM 2211 88.9 IRX4204 + Lapatinib 74396.3 Control 6696 IRX4204 1000 nM 5218 22.1 If Additive = 89.5%Neratinib 1000 nM 903 86.5 IRX4204 + Neratinib 329 95.1

TABLE 3 Synergistic Effect of the Combination of IRX4204 and Lapatinibor Neratinib on the Inhibition of the Growth of BT474 Cells. DAPI NucleiTreatment Count % Inhibition Control 2937 IRX4204 1000 nM 3399 −15.7* IfAdditive = 58.8% Lapatinib 1000 nM 1211 58.8 IRX4204 + Lapatinib 81372.3 Control 2192 IRX4204 1000 nM 2043 6.8 If Additive = 92.4% Neratinib1000 nM 180 91.8 IRX4204 + Neratinib 130 94.1 *Zero inhibition used incalculation of predicted additive effect

TABLE 4 Synergistic Effect of the Combination of IRX4204 and Lapatinibor Neratinib on the Inhibition of the Growth of MDA-MB-361 Cells. DAPINuclei Treatment Count % Inhibition Control 6696 IRX4204 1000 nM 521822.1 If Additive = 89.5% Lapatinib 1000 nM 903 86.5 IRX4204 + Lapatinib329 95.2 Control 5297 IRX4204 1000 nM 2459 43.6 If Additive = 97.3%Neratinib 1000 nM 249 95.3 IRX4204 + Neratinib 57 98.9

In each case the observed degree of inhibition exceeded that predictedif there was no interaction between the effects of the two agents, eventhough in some cases the individual therapeutic agents were quiteeffective alone, leaving little room for synergy to be observed. Thesedata also clearly indicate that IRX4204 and the small molecule Her2kinase inhibitors interact in a synergistic manner.

Example 3 Inhibition of Growth of a Her2-Targeted Therapeutic AgentResistant Her2⁺ Cancer Cell Line

HCC1954 is Her2⁺ cancer cell line derived from a ductal carcinoma of thebreast. As compared to the SkBr3 cell line used in Example 1 above, itis resistant to neratinib, lapatinib, and tucatinib. It is alsoresistant to anti-Her2 monoclonal antibodies, such as trastuzumab.Additionally, it is highly resistant to IRX4204. Among Her2-amplifiedcell lines, HCC1954 is the most resistant to neratinib with an IC50>100nM (compared to 7 nM SkBr3 or 20 nM AU565).

HCC1954 human breast cancer cells were seeded at 3000 cells per well in96-well plates and treated with neratinib [1 or 10 nM] or DMSO (0.1%) incombination with IRX4204 [10, 100, or 1000 nM]. After 6 days oftreatment, cells were fixed with 4% paraformaldehyde. Nuclei werestained with DAPI, imaged using ImageXpress® Pico (Molecular Devices)and counted using CellReporterXpress® Analysis Software. (FIGS. 4A-B)Each data point represents six replicates. Statistical significance wasdetermined using the Bonferroni-Dunn method of multiple t-tests. Eachday was analyzed individually, without assuming a consistent SD(*p<0.01). As IRX4204 had no growth inhibitory effect on this cell lineas a sole agent, the improved inhibition when used in combination withneratinib is a clear synergistic effect.

The experiment was also conducted using tucatinib or lapatinib in placeof neratinib. Tucatinib has an IC50>2 μM on HCC1954 as compared to 22 nMon SkBr3 or 125 nM on AU565. Lapatinib has an IC50≥1.4 μM on HCC1954 ascompared to 152 nM on SkBr3 or 294 nM on AU565. The same general patternof inhibitory effect is observed as with neratinib. Namely, the IRX4204had no growth inhibitory effect on this resistant cell line, the kinaseinhibitor has some small effect, but at the higher concentrationstested, the combination of the RXR agonist and the Her2 kinase inhibitorhad a profound growth inhibitory effect (FIG. 5A-B). As with neratinib,there is clearly a synergistic effect from the combination of thesekinase inhibitors with IRX4204.

While there are some common elements in the structures of these threekinase inhibitors, they are not close structural analogs. Thus, thesynergy observed between these three kinase inhibitors and IRX4204supports the generality of the effect with respect to Her2 kinaseinhibitors.

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe invention are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein is intended merely to betterilluminate the present invention and does not pose a limitation on thescope of the invention otherwise claimed. No language in the presentspecification should be construed as indicating any non-claimed elementessential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

1. A method of treating a patient with Her2⁺ cancer resistant to atleast one Her2-targeted therapeutic agent, the method comprisingadministering a retinoid X receptor (RXR) agonist of Formula I

wherein R is H, or lower alkyl of 1 to 6 carbons, or apharmaceutically-acceptable salt thereof, to the patient, wherein thepatient has received or is receiving the Her2-targeted therapeuticagent.
 2. The method of claim 1, further comprising administering theHer2-targeted therapeutic agent.
 3. The method of claim 1, furthercomprising administering thyroid hormone in conjunction with the RXRagonist.
 4. The method of claim 1, wherein the RXR agonist is a compoundof Formula II


5. The method of claim 1, wherein the Her2-targeted therapeutic agentcomprises an anti-Her2 therapeutic antibody.
 6. The method of claim 5,wherein the therapeutic antibody is trastuzumab or pertuzumab.
 7. Themethod of claim 1, wherein the Her2-targeted therapeutic agent comprisesan antibody-drug conjugate wherein the antibody is an anti-Her2antibody.
 8. The method of claim 1, wherein the Her2-targetedtherapeutic agent comprises a Her2 kinase inhibitor.
 9. The method ofclaim 8, wherein the Her2 kinase inhibitor is lapatinib, neratinib ortucatinib.
 10. The method of claim 8, wherein the Her2 kinase inhibitorcomprises afatinib, dacomitinib, canertinib, sapitinib, CP-724714, orCUDC-101.
 11. The method of claim 1, wherein the Her2⁺ cancer is Her2⁺breast cancer.
 12. The method of claim 1, wherein the Her2⁺ cancerresistant to a Her2-targeted therapeutic agent is also resistant to theRXR agonist of Formula I.
 13. The method of claim 1, wherein tumor cellgrowth is inhibited by a combination of the Her2-targeted therapeuticand the RXR agonist, and the inhibition of tumor cell growth by thecombination is greater than additive inhibitory effects of each of theHer2-targeted therapeutic and the RXR agonist alone.
 14. A method oftreating a patient with Her2⁺ cancer undergoing treatment with aHer2-targeted therapeutic agent, wherein the cancer has become resistantto the Her2-targeted therapeutic agent, the method comprising continuingtreatment with the Her2-targeted therapeutic agent and initiatingtreatment with an RXR agonist of Formula I,

wherein R is H, or lower alkyl of 1 to 6 carbons, or apharmaceutically-acceptable salt thereof.
 15. The method of claim 14,further comprising administering thyroid hormone in conjunction with theRXR agonist.
 16. The method of claim 14, wherein the RXR agonist is acompound of Formula II


17. The method of claim 14, wherein the Her2-targeted therapeutic agentcomprises an anti-Her2 therapeutic antibody.
 18. The method of claim 17,wherein the therapeutic antibody is trastuzumab or pertuzumab.
 19. Themethod of claim 14, wherein the Her2-targeted therapeutic agentcomprises an antibody-drug conjugate wherein the antibody is ananti-Her2 antibody.
 20. The method of claim 14, wherein theHer2-targeted therapeutic agent comprises a Her2 kinase inhibitor. 21.The method of claim 20, wherein the Her2 kinase inhibitor is lapatinib,neratinib or tucatinib.
 22. The method of claim 20, wherein the Her2kinase inhibitor comprises afatinib, dacomitinib, canertinib, sapitinib,CP-724714, or CUDC-101.
 23. The method of claim 14, wherein the Her2⁺cancer is Her2⁺ breast cancer.
 24. The method of claim 14, wherein theHer2⁺ cancer resistant to a Her2-targeted therapeutic agent is alsoresistant to the RXR agonist of Formula I.
 25. The method of claim 14,wherein tumor cell growth is inhibited by a combination of theHer2-targeted therapeutic and the RXR agonist, and the inhibition oftumor cell growth by the combination is greater than additive inhibitoryeffects of each of the Her2-targeted therapeutic and the RXR agonistalone.